Intranasal immunization with a formalin-inactivated human influenza A virus whole-virion vaccine alone and intranasal immunization with a split-virion vaccine with mucosal adjuvants show similar levels of cross-protection

Abstract

The antigenicity of seasonal human influenza virus changes continuously; thus, a cross-protective influenza vaccine design needs to be established. Intranasal immunization with an influenza split-virion (SV) vaccine and a mucosal adjuvant induces cross-protection; however, no mucosal adjuvant has been assessed clinically. Formalin-inactivated intact human and avian viruses alone (without adjuvant) induce cross-protection against the highly pathogenic H5N1 avian influenza virus. However, it is unknown whether seasonal human influenza formalin-inactivated whole-virion (WV) vaccine alone induces cross-protection against strains within a subtype or in a different subtype of human influenza virus. Furthermore, there are few reports comparing the cross-protective efficacy of the WV vaccine and SV vaccine-mucosal adjuvant mixtures. Here, we found that the intranasal human influenza WV vaccine alone induced both the innate immune response and acquired immune response, resulting in cross-protection against drift variants within a subtype of human influenza virus. The cross-protective efficacy conferred by the WV vaccine in intranasally immunized mice was almost the same as that conferred by a mixture of SV vaccine and adjuvants. The level of cross-protective efficacy was correlated with the cross-reactive neutralizing antibody titer in the nasal wash and bronchoalveolar fluids. However, neither the SV vaccine with adjuvant nor the WV vaccine induced cross-reactive virus-specific cytotoxic T-lymphocyte activity. These results suggest that the intranasal human WV vaccine injection alone is effective against variants within a virus subtype, mainly through a humoral immune response, and that the cross-protection elicited by the WV vaccine and the SV vaccine plus mucosal adjuvants is similar.

Fig 1

Cross-protection of mice against the homotype or subtype influenza virus strains by intranasal immunization with A/PR/8/34 vaccines. Groups of mice (5 per group) were inoculated twice intranasally with PBS, 100 μg of γ-PGA-NPs (NP), a mixture of 1 μg of A/PR/8/34 SV vaccine and 100 μg of γ-PGA-NPs (SV+NP), or 1 μg of A/PR/8/34 WV vaccine (WV). Fourteen days after the final immunization, the mice were infected with A/PR/8/34 (H1N1; 10³ PFU) (A), A/Hiroshima/53/2005 (H3N2; 10⁷ PFU) (B), or B/Malaysia/2506/2004 (10⁶ PFU) (C), and the mortality was assessed. *, P < 0.05 versus the group inoculated intranasally with PBS.

Fig 2

Cross-protection of mice against drift variants within a viral subtype by intranasal immunization with A/Solomon Islands/3/2006 vaccines. Groups of mice (5 per group) were inoculated twice intranasally with PBS, 100 μg of γ-PGA-NPs (NP), a mixture of 1 μg of A/Solomon Islands/3/2006 SV vaccine and 100 μg of γ-PGA-NPs (SV+NP), or 1 μg of A/Solomon Islands/3/2006 WV vaccine (WV). Fourteen days after the final immunization, the mice were infected with A/Solomon Islands/3/2006 (H1N1; 10⁷ PFU) (A), A/Brisbane/59/2007 (H1N1; 10⁷ PFU) (B), A/New Caledonia/20/99 (H1N1; 10⁷ PFU) (C), or A/PR/8/34 (H1N1; 10³ PFU) (D), and the mortality was assessed. *, P < 0.05 versus the group inoculated intranasally with PBS.

Fig 3

Cross-protection of mice against drift variants within a viral subtype by subcutaneous immunization with A/Solomon Islands/3/2006 vaccines. Groups of mice (5 per group) were inoculated twice subcutaneously with PBS, 100 μg of γ-PGA-NPs (NP), a mixture of 1 μg of A/Solomon Islands/3/2006 SV vaccine and 100 μg of γ-PGA-NPs (SV+NP), or 1 μg of A/Solomon Islands/3/2006 WV vaccine (WV). Fourteen days after the final immunization, the mice were infected with A/Solomon Islands/3/2006 (H1N1; 10⁷ PFU) (A), A/Brisbane/59/2007 (H1N1; 10⁷ PFU) (B), A/New Caledonia/20/99 (H1N1; 10⁷ PFU) (C), or A/PR/8/34 (H1N1; 10³ PFU) (D), and the mortality was assessed. *, P < 0.05 versus the group inoculated intranasally with PBS.

Fig 4

Cross-protection of mice against the homotype, a drift variant within the subtype, or different subtypes of an influenza virus strain by intranasal immunization with A/PR/8/34 WV vaccine and its SV vaccine plus poly(I·C). Groups of mice (5 per group) were inoculated twice intranasally with PBS, 10 μg of poly(I·C), a mixture of 1 μg of A/PR/8/34 SV vaccine and 10 μg of poly(I·C) [SV+poly(I·C)], or 1 μg of A/PR/8/34 WV vaccine (WV). Fourteen days after the final immunization, the mice were infected with A/PR/8/34 (H1N1; 10³ PFU) (A), A/Brisbane/59/2007 (H1N1; 10⁷ PFU) (B), or A/Hiroshima/53/2005 (H3N2; 10⁷ PFU) (C), and the mortality was assessed. *, P < 0.05 versus the group inoculated intranasally with PBS.

Fig 5

In vivo clearance of homotype and subtype of influenza virus strains by intranasal immunization of mice with A/PR/8/34 vaccines. Groups of mice (5 per group) were inoculated intranasally with PBS, a mixture of 1 μg of A/PR/8/34 SV vaccine and 100 μg of γ-PGA-NPs (SV+NP), or 1 μg of A/PR/8/34 WV vaccine (WV) as described in Materials and Methods. Fourteen days after the final immunization, the mice were infected with 10⁴ or 10⁵ PFU of A/PR/8/34 (H1N1), A/Hiroshima/53/2005 (H3N2), or B/Malaysia/2506/2004. Two days after the infection, the BAL fluid was harvested and the number of PFU of A/PR/8/34 (A), A/Hiroshima/52/2005 (B), or B/Malaysia/2506/2004 (C) was assessed. Bars represent the means ± standard deviations from 5 mice. *, P < 0.05 versus the group of mice inoculated intranasally with PBS.

Fig 6

In vivo clearance of H1N1 variants of influenza virus by the intranasal immunization of mice with A/Solomon Islands/3/2006 vaccines. Groups of mice (5 per group) were inoculated intranasally with PBS (PBS i.n.), a mixture of 1 μg A/Solomon Islands/3/2006 SV vaccine and 100 μg of γ-PGA-NPs (SV+NP i.n.), or 1 μg of A/Solomon Islands/3/2006 WV vaccine (WV i.n.), as described in Materials and Methods. Other groups of mice (5 per group) were immunized subcutaneously with PBS (PBS s.c.) or 1 μg of A/Solomon Islands/3/2006 WV vaccine (WV s.c.) as described in Materials and Methods. Fourteen days after the final immunization, the mice were infected with 10⁴ or 10⁵ PFU of A/Solomon Islands/3/2006, A/Brisbane/59/2007, A/New Caledonia/20/99, or A/PR/8/34. Two days after the infection, the BAL fluid was harvested and the PFU value for A/Solomon islands (A), A/Brisbane/59/2007 (B), A/New Caledonia/20/99 (C), or APR/8/34 (D) was assessed. Bars represent the means ± standard deviations from 5 mice. *, P < 0.05 versus the group of mice inoculated intranasally with PBS. #, P < 0.05 versus the group of mice inoculated subcutaneously with PBS.

Fig 7

Expression of cytokine mRNAs in the NALTs and cervical lymph nodes. Total RNA was extracted from the NALTs and cervical lymph nodes of mice intranasally treated with PBS, 1 μg of γ-PGA-NPs (NP), 1 μg of SV vaccine (SV), SV vaccine with γ-PGA-NPs (SV+NP), or WV vaccine (WV) from A/PR/8/34 and A/Solomon Islands/3/2006. The mRNA levels of IFN-α in the NALTs (A) and of IFN-β, IFN-γ, IL-4, IL-6, and IL-12 p40 in the cervical lymph nodes (B) were determined by real-time RT-PCR (n = 3). *, P < 0.05 for WV compared to PBS; **, P < 0.05 for SV+NP compared to NP; #, P < 0.05 for A/PR/8/34 SV+NP compared to A/Solomon Islands/3/2006 SV+NP; ##, P < 0.05 for A/PR/8/34 WV compared to A/Solomon Islands/3/2006 WV.

Fig 8

Cross-reactive anti-influenza neutralizing antibody titer in the nasal wash and BAL fluids of mice intranasally immunized with A/PR/8/34 vaccines. (A) Groups of mice (50 per group) were inoculated twice intranasally with PBS, 100 μg of γ-PGA-NPs (NP), a mixture of 1 μg of A/PR/8/34 SV vaccine and 100 μg of γ-PGA-NPs (SV+NP), or 1 μg of A/PR/8/34 WV vaccine (WV). Fourteen days after the final immunization, the BAL and nasal wash fluids were harvested. Fluids from 5 mice were pooled, pretreated with RDE, and concentrated. The neutralizing antibody titers for A/PR/8/34 (H1N1), A/Hiroshima/52/2005 (H3N2), and B/Malaysia/2506/2005 in the concentrated samples were assessed. Bars represent the means ± standard deviations from 10 concentrated samples. (B) Anti-influenza neutralizing antibody titer for drift variants within a virus subtype in the nasal wash and BAL fluids of mice intranasally immunized with A/Solomon Islands/3/2006 vaccines. Groups of mice (50 per group) were inoculated twice intranasally with PBS, 100 μg of γ-PGA-NPs (NP), a mixture of 1 μg of A/Solomon islands/3/2006 SV vaccine and 100 μg of γ-PGA-NPs (SV+NP), or 1 μg of A/Solomon Islands/3/2006 WV vaccine (WV). Fourteen days after the final immunization, the BAL and nasal wash fluids were harvested. Fluids from 5 mice were pooled, pretreated with RDE, and concentrated. The neutralizing antibody titers for A/Solomon Islands/3/2006, A/Brisbane/59/2007, A/New Caledonia/20/99, and A/PR/8/34 were then assessed. Bars represent the means ± standard deviations from 10 concentrated samples.

Fig 9

Cross-reactive anti-influenza CTL activity in intranasally immunized mice. (A) Mice (5 mice per group) were injected intranasally twice with PBS, a mixture of 1 μg of A/PR/8/34 SV vaccine and 100 μg of γ-PGA-NPs (SV+NP), or 1 μg of A/PR/8/34 WV vaccine (WV) on days 0 and 21, or they were infected with A/PR/8/34 (10 PFU; Virus) once on day 0. The mice then were challenged with A/PR/8/34 (10² PFU), A/Hiroshima/52/2005 (10⁵ PFU), or B/Malaysia/2506/2004 (10⁵ PFU) on day 35. Lymphocytes in the lungs were harvested 5 days postchallenge, and the lymphocytes (effector cells) were mixed with ⁵¹Cr-labeled P815 cells infected with the challenged influenza virus (target cells) at an effector cell-to-target cell ratio of 50:1. Following a 5-h incubation at 37°C, 5% CO₂, 100 μl of the supernatant was examined for the concentration of ⁵¹Cr released. (B) Mice were injected twice intranasally with PBS, a mixture of 1 μg of A/Solomon Islands/3/2006 SV vaccine and 100 μg of γ-PGA-NPs (SV+NP), or 1 μg of A/Solomon islands/3/2006 WV vaccine (WV) on days 0 and 21, or they were infected with A/Solomon Islands/3/2006 (10⁵ PFU; Virus) once on day 0. The mice were challenged with A/Solomon Islands/3/2006 (10³ PFU), A/Brisbane/59/2007 (10⁵ PFU), A/New Caledonia/20/99 (10⁵ PFU), or A/PR/8/34 (10² PFU) on day 42. Lymphocytes in the lungs were harvested 5 days postchallenge, pooled, and mixed with ⁵¹Cr-labeled P815 cells infected with the challenged influenza virus, and the CTL assay was performed as described for panel A. *, P < 0.05 versus the group of mice immunized with PBS.